M3R stimulates endocytosis of two variants of SLC4A4 in rat ParC5 cell

Abstract

Salivary ion and fluid secretion is regulated by parasympathetic activity mediated through muscarinic M3R receptors in acinar cells. The SLC4 bicarbonate transporters are the major passages for salivary bicarbonate secretion. We previously reported that in Xenopus oocytes, AT1 signaling regulates NBC1 function and surface expression by endocytosis. Here we study M3R signaling regulation of endogenous and recombinant NBC1 in rat parotid acinar cells (ParC5). We localized NBC1 to the basolateral membrane (BLM) in ParC5 cells. We showed that PMA and Carbachol induce redistribution of the cotransporter from the cell surface to endosomes. Inhibition of NBC1 recycling with the carboxylic ionophore monensin also caused significant accumulation of NBC1 in endosomes and a parallel loss of NBC1 from the cell surface. Co‐localization of internalized endogenous NBC1, NBC1‐A‐YFP or NBC1‐B‐GFP with Texas Red‐conjugated transferrin or Early Endosomal Antigen 1 antibody suggests clathrin‐dependent endocytosis within both contituitive and stimulated endocytosis. We showed that PMA and Carbachol induced PKC‐dependent endocytosis of NBC1 by using GF109203X. Using GÖ6976, we eliminated the involvement of PKCαβγ. We then identified that PKCδ is necessary in PMA and Carbachol stimulated endocytosis by knockdown of the endogenous PKCδ by expressing its kinase‐dead mutant. We have also reported that calmodulin antagonist W13 inhibits constitutive recycling of NBC1‐A in Xenopus oocytes. Here we demonstrated that W13 blocks recycling of endogenous NBC1, NBC1‐A‐YFP and NBC1‐B‐GFP in ParC5 cells. Our data demonstrate that NBC1 is constitutively endocytosed through a calmodulin dependent mechanism and that PMA and M3R signaling induce PKCδ‐dependent endocytosis of NBC1‐A/B.